NO is a gaseous molecule formed in many mammalian cells and involved in a whole series of physiological and pathological processes. Thus, the endothelium-dependent relaxation of the vascular smooth muscles is due to NO.
The exact mechanism of the action of NO still is largely unknown. It is assumed that physiologic carrier substances play an important role.
As such possible carrier substances, i.a. S-nitrosated proteins are assumed (Stamler et al., PNAS 89 (1992), 444-448). Thus, several thiol group-containing proteins of the most varying nature and function have been nitrosated, such as, e.g. serum albumin, t-PA and kathepsin B (Stamler et al., Hallstrom et al. (in: Shock, Sepsis, and Organ Failure--Nitric oxide, Fourth Wiggers Bernard Conference (1994), pp. 310-321)).
For this, bovine serum albumin (BSA) nitrosation levels of 85% (Stamler et al.,) or of 90 to 95%, respectively, for human albumin (Hallstrom et al.) have been reported. It has, however, been shown that with the methodology applied in these publications for determining the S nitroso-binding not only to S-nitrosations, but also other nitrosations, such as, e.g., N-nitrosations to tryptophane residues, or C-nitrosations (to tyrosine) can be detected.
In particular, the spectroscopic measurement at 335 nm and a molar extinction coefficient of 3869 mol.sup.-1 cm.sup.-1 proved to be non-specific for the S-nitrosothiol binding. In a publication following upon Stamler et al, the same group could prove by reliable detection methods i.a. that in the produced nitrosated BSA-preparations the free SH groups on Cysteine 34 were nitrosated to 37% at the most (Zhang et al., Journal of Biological Chemistry 271 (24), 1996, 14271-14279).
This level also matches the known fact that in preparations of proteins having potentially free thiol groups, only 20 to 35% are, in fact, present in free, reduced SH form. Particularly in protein preparations derived from blood or which are contacted with plasma or plasma derivatives in the course of their preparation procedure, the remaining 65 to 80% are blocked, mostly by mixed S-S bonds with small, thiol-carrying compounds, e.g. free L-cysteine or glutathione, respectively (Katachalski et al., J. Am. Chem. Soc. 79 (1957), 4096-4099, DeMaster et al., Biochemistry 34 (1995), 11494-11499).
As regards the sulphur-containing groupings in the respective proteins, basically it must be differentiated between those present in tightly bonded or associated form, e.g. as intramolecular saturated disulfide bonds and which are decisive for the conformation of the proteins, and those representing the potentially free thiol group(s). The latter are a known parameter for the respective protein. Human serum albumin, e.g., in its native state has a single potentially free thiol group per molecule, i.e. cysteine at position 34. These potentially free thiol groups, however, tend to form intermolecular disulfides, and therefore they are also termed mixed disulfides. In plasma, up to 80% of these thiol groups are present as mixed disulfides and thus are not directly available as free thiol groups.
In tests, in which a preparation of human serum albumin wherein only 20% of cysteine-34 was present in the reduced, and thus free, form, was treated with dithiothreitol (DTT) prior to nitrosation so as to reduce the mixed disulfides of the potentially free SH group, various nitrosation levels could be attained in dependence on the nitrosation agents and reaction conditions (DeMaster et al.).
There is a problem with the reduction of potentially free thiol groups to free reactive thiol groups, if in addition also the conformation-determining disulfide bonds are broken up and thus the nativity of the proteins is lost. A further problem occurs in proteins or protein solutions which have previously been subjected to a treatment for inactivating possibly present pathogens, e.g. a heat treatment. It is then that a tendency to increased aggregate formation is observed.